Vaccines for use in treating various diseases and disorders

ABSTRACT

Various diseases and disorders associated with cellular senescence may be treated by immunizing a subject in need thereof against AGE-modified proteins or peptides of a cell. Immunizing a subject includes administering a vaccine that comprises an AGE antigen. Vaccines against AGE-modified proteins or peptides contain an AGE antigen, an adjuvant, optional preservatives and optional excipients.

BACKGROUND

Sarcopenia is the loss of muscle mass, quality and strength associatedwith aging. Humans begin to lose muscle mass and function at some pointin the third decade of life. This loss of muscle mass typicallyaccelerates around age 75. Sarcopenia develops in both physically activeand physically inactive people. As the average human lifespan continuesto increase, sarcopenia is becoming a significant health concern. Theloss of muscle mass from sarcopenia may lead to poor balance, reducedgait speed and frailty. Individuals suffering from sarcopenia are moresusceptible to injury and disability, and may be unable to liveindependently as a result. The spread of sarcopenia will likely resultin increases in health care and assisted living expenses.

Sarcopenia has been considered to be an inevitable result of aging andthe natural deterioration of the body over time. The primary treatmentfor sarcopenia is exercise. Physical exercise, particularly resistancetraining or strength training, can reduce the impact of sarcopenia.Testosterone, anabolic steroids, ghrelin, vitamin D, angiotensinconverting enzyme inhibitors (ACE inhibitors), eicosapentaenoic acid(EPA), myostatin, selective androgen receptor modulators (SARMs),urocortin II (Ucn2) and hormone replacement therapy have beeninvestigated or are being studied as potential treatments forsarcopenia. Despite this research, there are currently no U.S. Food andDrug Administration (FDA)-approved agents for treating sarcopenia.

A recent study has identified a causal link between cellular senescenceand age-related disorders, such as sarcopenia. A research team at theMayo Clinic in Rochester, Minn., demonstrated that effects of aging inmice could be delayed by eliminating senescent cells in their fat andmuscle tissues without overt side effects (Baker, D. J. et al.,“Clearance of p16^(ink4a)-positive senescent cells delaysageing-associated disorders”, Nature, Vol. 479, pp. 232-236, (2011)).Elimination of senescent cells in transgenic mice was shown tosubstantially delay the onset of sarcopenia and cataracts, and to reducesenescence indicators in skeletal muscle and the eye. The studyestablished that life-long and late-life treatment of transgenic micefor removal of senescent cells has no negative side effects andselectively delays age-related phenotypes that depend on cells (Id.,page 234, col. 2, line 16 through page 235, col. 1, line 2). The authorstheorized that removal of senescent cells may represent an avenue fortreating or delaying age-related diseases in humans and improvinghealthy human lifespan (Id., page 235, col. 2, lines 38-51).

Senescent cells are cells that are partially-functional ornon-functional and are in a state of irreversible proliferative arrest.Senescence is a distinct state of a cell, and is associated withbiomarkers, such as activation of the biomarker p16^(ink4a), andexpression of β-galactosidase. Senescence begins with damage or stress(such as overstimulation by growth factors) of cells.

Advanced glycation end-products (AGEs; also referred to AGE-modifiedproteins, or glycation end-products) arise from a non-enzymatic reactionof sugars with protein side-chains in aging cells (Ando, K. et al.,Membrane Proteins of Human Erythrocytes Are Modified by AdvancedGlycation End Products during Aging in the Circulation, Biochem BiophysRes Commun., Vol. 258, 123, 125 (1999)). This process begins with areversible reaction between the reducing sugar and the amino group toform a Schiff base, which proceeds to form a covalently-bonded Amadorirearrangement product. Once formed, the Amadori product undergoesfurther rearrangement to produce AGEs. Hyperglycemia, caused by diabetesmellitus (DM), and oxidative stress promote this post-translationalmodification of membrane proteins (Lindsey JB, et al., “Receptor ForAdvanced Glycation End-Products (RAGE) and soluble RAGE (sRAGE):Cardiovascular Implications,” Diabetes Vascular Disease Research, Vol.6(1), 7-14, (2009)). AGEs may also be formed from other processes. Forexample, the advanced glycation end product,N^(ε)-(carboxymethyl)lysine, is a product of both lipid peroxidation andglycoxidation reactions. AGEs have been associated with severalpathological conditions including diabetic complications, inflammation,retinopathy, nephropathy, atherosclerosis, stroke, endothelial celldysfunction, and neurodegenerative disorders (Bierhaus A, “AGEs andtheir interaction with AGE-receptors in vascular disease and diabetesmellitus. I. The AGE concept,” Cardiovasc Res, Vol. 37(3), 586-600(1998)).

AGE-modified proteins are also a marker of senescent cells. Thisassociation between glycation end-product and senescence is well knownin the art. See, for example, Gruber, L. (WO 2009/143411, 26 Nov. 2009),Ando, K. et al. (Membrane Proteins of Human Erythrocytes Are Modified byAdvanced Glycation End Products during Aging in the Circulation, BiochemBiophys Res Commun., Vol. 258, 123, 125 (1999)), Ahmed, E.K. et al.(“Protein Modification and Replicative Senescence of WI-38 HumanEmbryonic Fibroblasts” Aging Cells, vol. 9, 252, 260 (2010)), Vlassara,H. et al. (Advanced Glycosylation Endproducts on Erythrocyte CellSurface Induce Receptor-Mediated Phagocytosis by Macrophages, J. Exp.Med., Vol. 166, 539, 545 (1987)) and Vlassara et al.(“High-affinity-receptor-mediated Uptake and Degradation ofGlucose-modified Proteins: A Potential Mechanism for the Removal ofSenescent Macromolecules” Proc. Nat!. Acad. Sci. USAl , Vol. 82, 5588,5591 (1985)). Furthermore, Ahmed, E.K. etal. indicates that glycationend-products are “one of the major causes of spontaneous damage tocellular and extracellular proteins” (Ahmed, E.K. et al., see above,page 353). Accordingly, the accumulation of glycation end-products isassociated with senescence and lack of function.

Cellular senescence and the accumulation of AGEs have been implicated ina number of diseases and disorders in addition to sarcopenia andage-related disorders. Senescence of cells in the central nervous systemsuch as glial cells, astrocytes and microglial cells has been associatedwith neurodegenerative disorders. Abnormal accumulation of senescentastrocytes has been associated with Alzheimer's disease (AD) (Bhat, R.et al., “Astrocyte Senescence as a Component of Alzheimer's Disease”,PLOS ONE, Vol. 7(9), e45069, pp. 1-10 (September 2012)). Microglial cellsenescence associated with normal aging is exacerbated by the presenceof the amyloid plaques indicative of AD (Flanary, B. E. et al.,“Evidence That Aging And Amyloid Promote Microglial Cell Senescence”,Rejuvenation Research, Vol. 10(1), pp. 61-74 (March 2007)). The presenceof AGEs with astrocytes and microglial cells in AD is further evidenceof the presence of senescent cells (Takeda, A., et aL “Advancedglycation end products co-localize with astrocytes and microglial cellsin Alzheimer's disease brain”, Acta Neuropathologica, Vol. 95, pp.555-558 (1998)). On the basis of recently reported findings, Chinta etaL proposed that environmental stressors associated with Parkinson'sdisease (PD) may act in part by eliciting senescence within non-neuronalglial cells, contributing to the characteristic decline in neuronalintegrity that occurs in this disorder (Chinta, S. J. et al.“Environmental stress, ageing and glial cell senescence: a novelmechanistic link to Parkinson's disease?”, J Intern Med, Vol. 273, pp.429-436 (2013)). Astrocyte senescence is also associated with PD (M.Mori, “The Parkinsonian Brain: Cellular Senescence andNeurodegeneration, SAGE (Jun. 30, 2015)(sage.buckinstitute.org/the-parkinsonian-brain-cellular-senescence-and-neurodegeneration/).In a rodent model of familial amyotrophic lateral sclerosis (ALS)overexpressing mutant superoxide dismutase-1 (m-SOD1), the rate ofastrocytes acquiring a senescent phenotype is accelerated (Das, M. M.and Svendsen, C. N., “Astrocytes show reduced support of motor neuronswith aging that is accelerated in a rodent model of ALS”, Neurobiologyof Aging, Vol. 36, pp. 1130-1139 (2015)). Even in multiple sclerosis(MS), microglia and macrophages are shifted toward a stronglyproinflammatory phenotype, reminiscent of SASP, and may potentiateneuronal damage by releasing proinflammatory cytokines and molecules(Luessi, F., et aL “Neurodegeneration in multiple sclerosis: noveltreatment strategies” Expert Rev. Neurother., Vol 9, pp.1061-1077(2012)).

Some neurodegenerative disorders are associated with abnormal cellularsenescence outside the central nervous system. Most satellite cells,also known as myosatellite cells, present in the muscle tissue of ALSpatients exhibit an abnormal senescent-like morphology, although theymay be capable of proliferating in vitro (Pradat, P.-F. et al.,“Abnormalities of satellite cells function in amyotrophic lateralsclerosis” Amyotrophic Lateral Sclerosis, Vol. 12, pp. 264-271 (2011)).Satellite cells are small multipotent cells found in mature muscle,which are able to give rise to additional satellite cells, ordifferentiate into myoblasts as well as provide additional myonuclei. Inan animal model of Duchenne muscular dystrophy (MD), reducedproliferative capacity and premature senescence of myoblasts wasobserved (Wright, W. E., “Myoblast Senescence in Muscular Dystrophy” ExpCell Res, Vol. 157, pp. 343-354 (1985)). Myoblasts are precursor cellswhich differentiate into myocytes (also referred to as muscle cells).

Neurodegenerative disorders are also associated with abnormal proteinaccumulations (King, O.D., et al., “The tip of the iceberg: RNA-bindingproteins with prion-like domains in neurodegenerative disease” BrainRes. Vol.1462, pp. 61-80 (2012)). A characteristic of PD and Lewy bodydementia is the formation of Lewy bodies that form inside nerve cells.The primary structural component of the Lewy bodies is alpha-synucleinprotein, in the form of fibrils. The presence of tangles and plaques area characteristic of AD, the presence of which is used to definitivelydiagnose the condition. Plaques, composed of beta-amyloid protein (alsoreferred to as amyloid beta, Aβ or Abeta), accumulate between nervecells. Tangles, composed of tau protein, form twisted fibers withincells. Prion diseases (also known as transmissible spongiformencephalopathies (TSEs)), include a variety of human and animal disordersuch as Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob disease,bovine spongiform encephalopathy (“mad cow” disease), scrapie (in sheepand goats), chronic wasting disease (in deer and elk), kuru and fatalfamilial insomnia. Prion protein is a misfolded protein molecule whichmay propagate by transmitting a misfolded protein state, resulting inthe accumulation of the misfolded protein and causing tissue damage andcell death (Dobson, D. M., “The structural basis of protein folding andits links with human disease” Phil. Trans. R. Soc. Lond. B, Vol. 356,pp. 133-145 (2001)). In these diseases, it is believed the protein is anormal protein which misfolds or forms an abnormal aggregate. In thecase of some patients with familial ALS, a mutated superoxidedismutase-1 (SOD1) forms inclusions and accumulates (Kato, S., etal.“Advanced glycation endproduct-modified superoxide dismutase-1(SOD1)-positive inclusions are common to familial amyotrophic lateralsclerosis patients with SOD1 gene mutations and transgenic miceexpressing human SOD1 with a G85R mutation” Acta Neuropathol, Vol. 100,pp. 490-505 (2000)).

The damage or stress that causes cellular senescence also negativelyimpacts mitochondrial DNA in the cells to cause them to produce freeradicals which react with sugars in the cell to form methyl glyoxal(MG). MG in turn reacts with proteins or lipids to generate advancedglycation end products. In the case of the protein component lysine, MGreacts to form carboxymethyllysine, which is an AGE.

Damage or stress to mitochondrial DNA also sets off a DNA damageresponse which induces the cell to produce cell cycle blocking proteins.These blocking proteins prevent the cell from dividing. Continued damageor stress causes mTOR production, which in turn activates proteinsynthesis and inactivates protein breakdown. Further stimulation of thecells leads to programmed cell death (apoptosis). p16 is a proteininvolved in regulation of the cell cycle, by inhibiting the S phase. Itcan be activated during ageing or in response to various stresses, suchas DNA damage, oxidative stress or exposure to drugs. p16 is typicallyconsidered a tumor suppressor protein, causing a cell to becomesenescent in response to DNA damage and irreversibly preventing the cellfrom entering a hyperproliferative state. However, there has been someambiguity in this regard, as some tumors show overexpression of p16,while other show downregulated expression. Evidence suggests thatoverexpression of p16 is some tumors results from a defectiveretinoblastoma protein (“Rb”). p16 acts on Rb to inhibit the S phase,and Rb downregulates p16, creating negative feedback. Defective Rb failsto both inhibit the S phase and downregulate p16, thus resulting inoverexpression of p16 in hyperproliferating cells. Romagosa, C. et al.,p16^(ink4a) overexpression in cancer: a tumor suppressor gene associatedwith senescence and high-grade tumors, Oncogene, Vol. 30, 2087-2097(2011).

Senescent cells are also known to fuel the growth of cancer cells.Senescent cells are associated with secretion of many factors involvedin intercellular signaling, including pro-inflammatory factors;secretion of these factors has been termed the senescence-associatedsecretory phenotype, or SASP. One study showed that senescentmesenchymal stem cells promote proliferation and migration of breastcancer cells by the secretion of IL-6 (Di, G-h. et al. IL-6 Secretedfrom Senescent Mesenchymal Stem Cells Promotes Proliferation andmigration of Breast Cancer Cells, PLOS One, Vol. 9, 11, e113572 (2014)).Another study showed that senescent human fibroblasts increase thegrowth of tumors by the secretion of matrix metalloproteinase (Liu, D.et al. Senescent Human Fibroblasts Increase the Early Growth ofXenograft Tumors via Matrix Metalloproteinase Secretion, Cancer Res,Vol. 67, 3117-3126 (2007)).

Vaccines have been widely used since their introduction by Edward Jennerin the 1770s to confer immunity against a wide range of diseases andafflictions. Vaccine preparations contain a selected immunogenic agentcapable of stimulating immunity to an antigen. Typically, antigens areused as the immunogenic agent in vaccines, such as, for example,viruses, either killed or attenuated, and purified viral components.Antigens used in the production of cancer vaccines include, for example,tumor-associated carbohydrate antigens (TACAs), dendritic cells, wholecells and viral vectors. Different techniques are employed to producethe desired amount and type of antigen being sought. For example,pathogenic viruses are grown either in eggs or cells. Recombinant DNAtechnology is often utilized to generate attenuated viruses forvaccines.

Immunity is a long-term immune response, either cellular or humoral. Acellular immune response is activated when an antigen is presented,preferably with a co-stimulator to a T-cell which causes it todifferentiate and produce cytokines. The cells involved in thegeneration of the cellular immune response are two classes of T-helper(Th) cells, Th1 and Th2. Th1 cells stimulate B cells to producepredominantly antibodies of the IgG2A isotype, which activates thecomplement cascade and binds the Fc receptors of macrophages, while Th2cells stimulate B cells to produce IgG1 isotype antibodies in mice, IgG4isotype antibodies in humans, and IgE isotype antibodies. The human bodyalso contains “professional” antigen-presenting cells such as dendriticcells, macrophages, and B cells.

A humoral immune response is triggered when a B cell selectively bindsto an antigen and begins to proliferate, leading to the production of aclonal population of cells that produce antibodies that specificallyrecognize that antigen and which may differentiate intoantibody-secreting cells, referred to as plasma-cells or memory-B cells.Antibodies are molecules produced by B-cells that bind a specificantigen. The antigen-antibody complex triggers several responses, eithercell-mediated, for example by natural killers (NK) or macrophages, orserum-mediated, for example by activating the complement system, acomplex of several serum proteins that act sequentially in a cascadethat result in the lysis of the target cell.

Immunological adjuvants (also referred to simply as “adjuvants”) are thecomponent(s) of a vaccine which augment the immune response to theimmunogenic agent. Adjuvants function by attracting macrophages to theimmunogenic agent and then presenting the agent to the regional lymphnodes to initiate an effective antigenic response. Adjuvants may alsoact as carriers themselves for the immunogenic agent. Adjuvants mayinduce an inflammatory response, which may play an important role ininitiating the immune response. Adjuvants include mineral compounds suchas aluminum salts, oil emulsions, bacterial products, liposomes,immunostimulating complexes and squalene.

Other components of vaccines include pharmaceutically acceptableexcipients, preservatives, diluents and pH adjusters. A variety of thesecomponents of vaccines, as well as adjuvants, are described inwww.cdc.gov/vaccines/pubs/pinkbook/downloads/appendices/B/excipient-table-2.pdfand Vogel, F. R. et aL, “A compendium of vaccine adjuvants andexcipients”, Pharmaceutical Biotechnology, Vol. 6, pp. 141-228 (1995).

Vaccines may therefore be used to stimulate the production of antibodiesin the body and provide immunity against antigens. When an antigen isintroduced to a subject that has been vaccinated and developed immunityto that antigen, the immune system may destroy or remove cells thatexpress the antigen.

SUMMARY

In a first aspect, the invention is a method of treating or preventing adisease or disorder associated with cellular senescence comprisingimmunizing a subject in need thereof against AGE-modified proteins orpeptides of a cell.

In a second aspect, the invention is a method of treating a subject witha disease or disorder associated with cellular senescence comprisingadministering a first vaccine comprising a first AGE antigen andadministering a second vaccine comprising a second AGE antigen. Thesecond AGE antigen is different from the first AGE antigen.

In a third aspect, the invention is use of an AGE antigen for themanufacture of a medicament for treating or preventing a disease ordisorder associated with cellular senescence.

In a fourth aspect, the invention is a composition comprising an AGEantigen for use in treating or preventing a disease or disorderassociated with cellular senescence.

In a fifth aspect, the invention is a composition comprising an AGEantigen for use in treating sarcopenia.

In a sixth aspect, the invention is a composition comprising an AGEantigen for use in promoting tissue or organ regeneration.

In a seventh aspect, the invention is a composition comprising an AGEantigen for use in promoting regenerative processes or overcoming agingeffects.

In an eighth aspect, the invention is a composition comprising an AGEantigen for use in treating atherosclerosis.

In an ninth aspect, the invention is a composition comprising an AGEantigen for use in preventing or delaying the onset of cataracts.

In a tenth aspect, the invention is a composition comprising an AGEantigen for use in preventing or delaying the onset of loss of adiposetissue.

In an eleventh aspect, the invention is a composition comprising an AGEantigen for use in preventing or delaying the onset of lordokyphosis.

In a twelfth aspect, the invention is a composition comprising an AGEantigen for use in treating inflammation or auto-immune disorders.

In a thirteenth aspect, the invention is a composition comprising an AGEantigen for use in treating neurodegenerative disorders.

In a fourteenth aspect, the invention is a composition comprising an AGEantigen for use in treating cancer or cancer metastases.

In a fifteenth aspect, the invention is a composition comprising an AGEantigen for use in increasing health span.

In a sixteenth aspect, the invention is a method of reducing the numberof AGE-modified cells in a patient, comprising administering a vaccinecomprising an AGE antigen.

Definitions

The term “peptide” means a molecule composed of 2-50 amino acids.

The term “protein” means a molecule composed of more than 50 aminoacids.

The term “sarcopenia” means the syndrome characterized by the presenceof (1) low muscle mass and (2) low muscle function (low muscle strengthor reduced physical performance). Muscle mass may be measured by bodyimaging techniques, such as computed tomography scanning (CT scan),magnetic resonance imaging (MRI) or dual energy X-ray absorptiometry(DXA or DEXA); bioimpedance analysis (BIA); body potassium measurement,such as total body potassium (TBK) or partial body potassium (PBK); oranthropometric measurements, such as mid-upper arm circumference, skinfold thickness or calf circumference. Preferably, muscle mass ismeasured by CT scan, MRI or DXA. Muscle strength may be measured byhandgrip strength, knee flexion/extension or peak expiratory flow.Preferably, muscle strength is measured by handgrip strength. Physicalperformance may be measured by the Short Physical Performance Battery,gait speed measurement, timed get-up-and-go (TGUG) or the stair climbpower test. Preferably, physical performance is measured by gait speedmeasurement. A subject may be identified as having sarcopenia or in needof treatment if (1) the subject is at least 25 years old and (2) his orher measured muscle mass and measured muscle function are two standarddeviations or more below the mean value for healthy 25 year olds of thesame gender and no alternative pathology has been identified to accountfor the reduced muscle mass and reduced muscle function. Preferably, asubject being treated for sarcopenia is at least 40 years old. Morepreferably, a subject being treated for sarcopenia is at least 50 yearsold. Most preferably, a subject being treated for sarcopenia is at least60 years old. Alternatively, a subject may be identified as havingsarcopenia or in need of treatment if (1) his or her gait speed is lessthan 1.0 m/s across a 4 m course and (2) he or she has an objectivelymeasured low muscle mass, such as, for example, an appendicular massrelative to the square of height less than or equal to 7.23 kg/m² formale subjects or less than or equal to 5.67 kg/m² for female subjects(Fielding, R. A., et al., “Sarcopenia: an undiagnosed condition in olderadults. Current consensus definition: prevalence, etiology, andconsequences”, Journal of the American Medical Directors Association,Vol. 12(4), pp. 249-256 (May 2011).

The term “neurodegenerative disorder” means disorders which result inneurons loosing function and/or dying, in the central nervous systemincluding the brain. Such disorders included central nervous systemneurodegenerative disorders such as AD, PD, Lewy body dementia, MS,prion diseases (also known as transmissible spongiform encephalopathies(TSEs), including Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakobdisease, bovine spongiform encephalopathy (“mad cow” disease), scrapie(in sheep and goats), chronic wasting disease (in deer and elk), kuruand fatal familial insomnia), and ALS.

“Neurodegenerative proteins” are proteins which accumulate in a patienthaving a neurodegenerative disorders and which are associated with theneurodegenerative disorder. Examples include, beta-amyloid proteinplaques (associated with AD), tau protein tangles (associated with AD),mutated superoxide dismutase-1 (associated with ALS), prion proteinaggregates (associated with TSEs) and alpha-synuclein protein fibrils(associated with PD and Lewy Body dementia). A “neurodegenerativeprotein” is the form of the protein which accumulates during theneurodegenerative disorder, typically a mutant or mis-folded form.

The terms “advanced glycation end-product,” “AGE,” “AGE-modified proteinor peptide,” and “glycation end-product” refer to modified proteins orpeptides that are formed as the result of the reaction of sugars withprotein side chains that further rearrange and form irreversiblecross-links. This process begins with a reversible reaction between areducing sugar and an amino group to form a Schiff base, which proceedsto form a covalently-bonded Amadori rearrangement product. Once formed,the Amadori product undergoes further rearrangement to produce AGEs.AGE-modified proteins and antibodies to AGE-modified proteins aredescribed in U.S. Pat. No. 5,702,704 to Bucala (“Bucala”) and U.S. Pat.No. 6,380,165 to Al-Abed et al. (“Al-Abed”). Glycated proteins orpeptides that have not undergone the necessary rearrangement to formAGEs, such as N-deoxyfructosyllysine found on glycated albumin, are notAGEs. AGEs may be identified by the presence of AGE modifications (alsoreferred to as AGE epitopes or AGE moieties) such as2-(2-furoyI)-4(5)-(2-furanyl)-1H-imidazole (“FFI”);5-hydroxymethyl-1-alkylpyrrole-2-carbaldehyde (“Pyrraline”);1-alkyl-2-formyl-3,4-diglycosyl pyrrole (“AFGP”), a non-fluorescentmodel AGE; carboxymethyllysine; and pentosidine. ALI, another AGE, isdescribed in Al-Abed.

The term “AGE antigen” means a substance that elicits an immune responseagainst an AGE-modified protein or peptide of a cell. The immuneresponse against an AGE-modified protein or peptide of a cell does notinclude the production of antibodies to the non-AGE-modified protein orpeptide.

The term “AGE antibody” means an antibody specific for an AGE-modifiedprotein or peptide of a cell.

The term “senescent cell” means a cell which is in a state ofirreversible proliferative arrest and expresses one or more biomarkersof senescence, such as activation of p16^(ink4a) or expression ofβ-galactosidase. Also included are cells which express one or morebiomarkers of senescence, do not proliferate in vivo, but mayproliferate in vitro under certain conditions, such as some satellitecells found in the muscles of ALS patients.

The term “increasing health span” means reducing age-related phenotypes.

Age-related phenotypes include, for example, sarcopenia, cataracts, lossof adipose tissue and lordokyphosis.

DETAILED DESCRIPTION

The identification of a link between cellular senescence and sarcopeniaallows for new treatment possibilities. For example, if the immunogenicagent of a vaccine is an AGE-modified protein or peptide, the immunesystem of an immunized subject may kill or induce apoptosis in cellsexpressing the AGE-modified protein or peptide.

The present invention uses enhanced clearance of cells expressingAGE-modified proteins or peptides (AGE-modified cells) to treat orameliorate sarcopenia. Vaccination against AGE-modified proteins orpeptides of a cell produces the desired result of controlling thepresence of AGE-modified cells in a subject in need thereof. Thecontinuous and virtually ubiquitous surveillance exercised by the immunesystem in the body in response to a vaccination allows maintaining lowlevels of AGE-modified cells in the body. Vaccination againstAGE-modified proteins or peptides of a cell can help remove or killsenescent cells. The process of senescent cell removal or destructionallows vaccination against AGE-modified proteins or peptides of a cellto be used to treat sarcopenia.

Vaccination against AGE-modified proteins or peptides of a cell may alsobe used for increasing health span. Health span may be increased byreducing age-related phenotypes. The vaccine may be used, for example,to prevent or delay the onset of cataracts, lordokyphosis or loss ofadipose tissue.

Other diseases or disorders that are associated with cellular senescencemay also be treated or ameliorated by vaccination against AGE-modifiedproteins or peptides of a cell. For example, the vaccine may be used totreat neurodegenerative disorders, cancer, cancer metastases oratherosclerosis.

Vaccines against AGE-modified proteins or peptides contain an AGEantigen, an adjuvant, optional preservatives and optional excipients.Examples of AGE antigens include AGE-modified proteins or peptides suchas AGE-antithrombin III, AGE-calmodulin, AGE-insulin, AGE-ceruloplasmin,AGE-collagen, AGE-cathepsin B, AGE-albumin, AGE-crystallin,AGE-plasminogen activator, AGE-endothelial plasma membrane protein,AGE-aldehyde reductase, AGE-transferrin, AGE-fibrin, AGE-copper/zincSOD, AGE-apo B, AGE-fibronectin, AGE-pancreatic ribose, AGE-apo A-I andII, AGE-hemoglobin, AGE-Na⁺/K⁺-ATPase, AGE-plasminogen, AGE-myelin,AGE-lysozyme, AGE-immunoglobulin, AGE-red cell Glu transport protein,AGE-β-N-acetyl hexominase, AGE-apo E, AGE-red cell membrane protein,AGE-aldose reductase, AGE-ferritin, AGE-red cell spectrin, AGE-alcoholdehydrogenase, AGE-haptoglobin, AGE-tubulin, AGE-thyroid hormone,AGE-fibrinogen, AGE-132-microglobulin, AGE-sorbitol dehydrogenase,AGE-α₁-antitrypsin, AGE-carbonate dehydratase, AGE-RNAse, AGE-lowdensity lipoprotein, AGE-hexokinase, AGE-apo C-I, AGE-RNAse,AGE-hemoglobin such as AGE-human hemoglobin, AGE-albumin such asAGE-bovine serum albumin (AGE-BSA) and AGE-human serum albumin, AGE-lowdensity lipoprotein (AGE-LDL) and AGE-collagen IV. AGE-modified cells,such as AGE-modified erythrocytes, whole, lysed, or partially digested,may also be used as AGE antigens. Suitable AGE antigens also includeproteins or peptides that exhibit AGE modifications (also referred to asAGE epitopes or AGE moieties) such as carboxymethyllysine,carboxyethyllysine, pentosidine, pyrraline, FFI, AFGP and ALI. Furtherdetails of some of these AGE-modified proteins or peptides and theirpreparation are described in Bucala.

A particularly preferred AGE antigen is a protein or peptide thatexhibits a carboxymethyllysine AGE modification. Carboxymethyllysine(also known as CML, N(epsilon)-(carboxymethyl)lysine,N(6)-carboxymethyllysine, or 2-Amino-6-(carboxymethylamino)hexanoicacid) is found on proteins or peptides and lipids as a result ofoxidative stress and chemical glycation, and has been correlated withaging. CML-modified proteins or peptides are recognized by the receptorRAGE which is expressed on a variety of cells. CML has been well-studiedand CML-related products are commercially available. For example, CellBiolabs, Inc. sells CML-BSA antigens, CML polyclonal antibodies, CMLimmunoblot kits, and CML competitive ELISA kits(www.cellbiolabs.com/cml-assays).

AGE antigens may be conjugated to carrier proteins to enhance antibodyproduction in a subject. Antigens that are not sufficiently immunogenicalone may require a suitable carrier protein to stimulate a responsefrom the immune system. Examples of suitable carrier proteins includekeyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin,cholera toxin, labile enterotoxin, silica particles and soybean trypsininhibitor. Preferably, the carrier protein is KLH. KLH has beenextensively studied and has been identified as an effective carrierprotein in experimental cancer vaccines. A preferred AGE antigen-carrierprotein conjugate is CML-KLH.

Adjuvants include mineral compounds such as aluminum salts, oilemulsions, bacterial products, liposomes, immunostimulating complexesand squalene. Aluminum compounds are the most widely used adjuvants inhuman and veterinary vaccines. These aluminum compounds include aluminumsalts such as aluminum phosphate (AIPO₄) and aluminum hydroxide(Al(OH)₃) compounds, typically in the form of gels, and are genericallyreferred to in the field of vaccine immunological adjuvants as “alum.”Aluminum hydroxide is a poorly crystalline aluminum oxyhydroxide havingthe structure of the mineral boehmite. Aluminum phosphate is anamorphous aluminum hydroxyphosphate. Negatively charged species (forexample, negatively charged antigens) can absorb onto aluminum hydroxidegels at neutral pH, whereas positively charged species (for example,positively charged antigens) can absorb onto aluminum phosphate gels atneutral pH. It is believed that these aluminum compounds provide a depotof antigen at the site of administration, thereby providing a gradualand continuous release of antigen to stimulate antibody production.Aluminum compounds tend to more effectively stimulate a cellularresponse mediated by Th2, rather than Th1 cells.

Emulsion adjuvants include water-in-oil emulsions (for example, Freund'sadjuvants, such as killed mycobacteria in oil emulsion) and oil-in-wateremulsions (for example, MF-59). Emulsion adjuvants include animmunogenic component, for example squalene (MF-59) or mannide oleate(Incomplete Freund's Adjuvants), which can induce an elevated humoralresponse, increased T cell proliferation, cytotoxic lymphocytes andcell-mediated immunity.

Liposomal or vesicular adjuvants (including paucilamellar lipidvesicles) have lipophilic bilayer domains and an aqueous milieu whichcan be used to encapsulate and transport a variety of materials, forexample an antigen. Paucilamellar vesicles (for example, those describedin U.S. Pat. No. 6,387,373) can be prepared by mixing, under highpressure or shear conditions, a lipid phase comprising anon-phospholipid material (for example, an amphiphile surfactant; seeU.S. Pat. Nos. 4,217,344; 4,917,951; and 4,911,928), optionally asterol, and any water-immiscible oily material to be encapsulated in thevesicles (for example, an oil such as squalene oil and an oil-soluble oroil-suspended antigen); and an aqueous phase such as water, saline,buffer or any other aqueous solution used to hydrate the lipids.Liposomal or vesicular adjuvants are believed to promote contact of theantigen with immune cells, for example by fusion of the vesicle to theimmune cell membrane, and preferentially stimulate the Th1sub-population of T-helper cells.

Other types of adjuvants include Mycobacterium bovis bacillusCalmette-Guérin (BCG), quill-saponin and unmethylated CpG dinucleotides(CpG motifs). Additional adjuvants are described in U.S. PatentApplication Publication Pub. No. US 2010/0226932 (Sep. 9, 2010) andJiang, Z-H. etal. “Synthetic vaccines: the role of adjuvants in immunetargeting”, Current Medicinal Chemistry, Vol. 10(15), pp. 1423-39(2003). Preferable adjuvants include Freund's complete adjuvant andFreund's incomplete adjuvant.

The vaccine may optionally include one or more preservatives, such asantioxidants, antibacterial and antimicrobial agents, as well ascombinations thereof. Examples include benzethonium chloride,ethylenediamine-tetraacetic acid sodium (EDTA), thimerosal, phenol,2-phenoxyethanol, formaldehyde and formalin; antibacterial agents suchas amphotericin B, chlortetracycline, gentamicin, neomycin, polymyxin Band streptomycin; antimicrobial surfactants such as polyoxyethylene-9,10-nonyl phenol (Triton N-101, octoxynol-9), sodium deoxycholate andpolyoxyethylated octyl phenol (Triton X-l00). The production andpackaging of the vaccine may eliminate the need for a preservative. Forexample, a vaccine that has been sterilized and stored in a sealedcontainer may not require a preservative.

Other components of vaccines include pharmaceutically acceptableexcipients, such as stabilizers, thickening agents, toxin detoxifiers,diluents, pH adjusters, tonicity adjustors, surfactants, antifoamingagents, protein stabilizers, dyes and solvents. Examples of suchexcipients include hydrochloric acid, phosphate buffers, sodium acetate,sodium bicarbonate, sodium borate, sodium citrate, sodium hydroxide,potassium chloride, potassium chloride, sodium chloride,polydimethylsilozone, brilliant green, phenol red(phenolsulfon-phthalein), glycine, glycerin, sorbitol, histidine,monosodium glutamate, potassium glutamate, sucrose, urea, lactose,gelatin, sorbitol, polysorbate 20, polysorbate 80 and glutaraldehyde.

The vaccine may be provided in unit dosage form or in multidosage form,such as 2-100 or 2-10 doses. The unit dosages may be provided in a vialwith a septum, or in a syringe with or without a needle. The vaccine maybe administered intravenously, subdermally or intraperitoneally.Preferably, the vaccine is sterile.

[60] The vaccine may be administered one or more times, such as 1 to 10times, including 2, 3, 4, 5, 6, 7, 8 or 9 times, and may be administeredover a period of time ranging from 1 week to 1 year, 2-10 weeks or 2-10months. Furthermore, booster vaccinations may be desirable, over thecourse of 1 year to 20 years, including 2, 5, 10 and 15 years.

A subject that receives a vaccine for AGE-modified proteins or peptidesof a cell may be tested to determine if he or she has developed animmunity to the AGE-modified proteins or peptides. Suitable tests mayinclude blood tests for detecting the presence of an antibody, such asimmunoassays or antibody titers. Alternatively, an immunity toAGE-modified proteins or peptides may be determined by measuring changesin muscle mass over time. For example, a baseline muscle mass in asubject may be measured followed by administration of the vaccine forAGE-modified proteins or peptides of a cell. Immunity to AGE-modifiedproteins or peptides may be determined by periodically measuring musclemass in the subject and comparing the subsequent measurements to thebaseline measurement. A subject may be considered to have developed animmunity to AGE-modified proteins or peptides if he or she does notdemonstrate loss of muscle mass between subsequent measurements or overtime. Alternatively, the concentration and/or number of senescent cellsin fat or muscle tissue may also be monitored. Vaccination andsubsequent testing may be repeated until the desired therapeutic resultis achieved.

The vaccination process may be designed to provide immunity againstmultiple

AGE moieties. A single AGE antigen may induce the production of AGEantibodies which are capable of binding to multiple AGE moieties.Alternatively, the vaccine may contain multiple AGE antigens. Inaddition, a subject may receive multiple vaccines, where each vaccinecontains a different AGE antigen.

Any mammal that could develop sarcopenia or other diseases or disordersassociated with cellular senescence may be treated by the methods hereindescribed. Humans are a preferred mammal for treatment. Other mammalsthat may be treated include mice, rats, goats, sheep, cows, horses andcompanion animals, such as dogs or cats. A subject in need of treatmentmay be identified by the diagnosis of a disease or disorder that isknown to cause elevated levels of AGEs such as, for example, diabetes(both Type 1 and Type 2), or the presence of a pathological conditionassociated with AGEs such as, for example, atherosclerosis,inflammation, retinopathy, nephropathy, stroke, endothelial celldysfunction, neurodegenerative disorders or cancer. In addition,subjects may be identified for treatment based on their age. Forexample, a human over 75 years of age may be treated for sarcopenia,while a human under 30 years of age might not be identified as in needof treatment for sarcopenia. Alternatively, any of the mammals orsubjects identified above may be excluded from the patient population inneed of treatment for sarcopenia.

A human subject may be identified as having sarcopenia or in need oftreatment if (1) the subject is at least 25 years old and (2) his or hermeasured muscle mass and measured muscle function are two standarddeviations or more below the mean value for healthy 25 year olds of thesame gender and no alternative pathology has been identified to accountfor the reduced muscle mass and reduced muscle function. Preferably, asubject being treated for sarcopenia is at least 40 years old. Morepreferably, a subject being treated for sarcopenia is at least 50 yearsold. Most preferably, a subject being treated for sarcopenia is at least60 years old. Alternatively, a subject may be identified as havingsarcopenia or in need of treatment if (1) his or her gait speed is lessthan 1.0 m/s across a 4 m course and (2) he or she has an objectivelymeasured low muscle mass, such as, for example, an appendicular massrelative to the square of height less than or equal to 7.23 kg/m² formale subjects or less than or equal to 5.67 kg/m² for female subjects.

Any mammal that could develop neurodegenerative disorders may be treatedby the methods herein described. Humans are a preferred mammal fortreatment. Other mammals that may be treated include mice, rats, goats,sheep, cows, horses and companion animals, such as dogs or cats. Asubject in need of treatment may be identified by the diagnosis of aneurodegenerative disorder.

In the case of cancer, a mammal that could develop metastatic cancer maybe treated by the methods herein described. Humans are a preferredmammal for treatment. Other mammals that may be treated include mice,rats, goats, sheep, cows, horses and companion animals, such as dogs orcats. A subject in need of treatment may be identified by the diagnosisof a cancer. Cancers which are particularly subject to metastasisinclude lung cancer, melanoma, colon cancer, renal cell carcinoma,prostate cancer, cancer of the cervix, bladder cancer, rectal cancer,esophageal cancer, liver cancer, mouth and throat cancer, multiplemyeloma, ovarian cancer, and stomach cancer. Treatment may be ofpatients experiencing metastatic cancer. Treatment may also beadministered to patients who have cancer, but prior to any identifiedmetastasis, in order to prevent metastasis. A subject that receivesadministration of an anti-AGE antibody may be tested to determine if ithas been effective to treat the cancer by examining the patient for thespread of the cancer to different parts of the body, particularly inlymph nodes. Administration of antibody and subsequent testing may berepeated until the desired therapeutic result is achieved.

EXAMPLES Example 1 (Prophetic): An AGE-RNAse containing vaccine in ahuman subject.

[69] AGE-RNAse is prepared by incubating RNAse in a phosphate buffersolution containing 0.1-3 M glucose, glucose-6-phosphate, fructose orribose for 10-100 days. The AGE-RNAse solution is dialyzed and theprotein content is measured. Aluminum hydroxide or aluminum phosphate,as an adjuvant, is added to 100 pg of the AGE-RNAse. Formaldehyde orformalin is added as a preservative to the preparation. Ascorbic acid isadded as an antioxidant. The vaccine also includes phosphate buffer toadjust the pH and glycine as a protein stabilizer.

The composition is injected into a human subject subcutaneously. Thesubject's muscle mass is measured at'the time of injection to establisha baseline muscle mass value. The patient's muscle mass is measuredagain after one month. The one-month muscle mass value is compared tothe baseline value. Additional injections are performed and additionalmuscle mass measurements are taken every month until the muscle massmeasurement indicates no change, or an increase, from the baselinevalue.

Example 2 (Prophetic): Injection regimen for an AGE-RNAse containingvaccine in a human subject.

The same vaccine as described in Example 1 is injected into a humansubject. The titer of antibodies to AGE-RNAse is determined by ELISAafter two weeks. Additional injections are performed after three weeksand six weeks, respectively. Further titer determination is performedtwo weeks after each injection.

Example 3 (Prophetic): An AGE-hemoglobin containing vaccine in a humansubject.

AGE-hemoglobin is prepared by incubating human hemoglobin in a phosphatebuffer solution containing 0.1-3 M glucose, glucose-6-phosphate,fructose or ribose for 10-100 days. The AGE-hemoglobin solution isdialyzed and the protein content is measured. All vaccine components arethe same as in Example 1, except AGE-hemoglobin is substituted forAGE-RNAse.

Administration is carried out as in Example 1, or as in Example 2. Thenumber of senescent cells in the subject's adipose tissue is measured atthe time of injection to establish a baseline number of senescent cells.The number of senescent cells in the subject's adipose tissue ismeasured again two months after injection and is compared to thebaseline number of senescent cells. Additional injections are performedand additional senescent cell measurements are taken every two months todetermine if the number of senescent cells in adipose tissue isincreasing or decreasing, or if there is no change in the number ofsenescent cells in adipose tissue.

Example 4 (Prophetic): An AGE-human serum albumin containing vaccine ina human subject.

AGE-human serum albumin is prepared by incubating human serum albumin ina phosphate buffer solution containing 0.1-3 M glucose,glucose-6-phosphate, fructose or ribose for 10-100 days. The AGE-humanserum albumin solution is dialyzed and the protein content is measured.All vaccine components are the same as in Example 1, except AGE-humanserum albumin is substituted for AGE-RNAse. Administration is carriedout as in Example 1, or as in Example 2.

Example 5: In vivo study of the administration of anti-AGE antibody.

To examine the effects of an anti-AGE antibody, the antibody wasadministered to the aged CD1(lCR) mouse (Charles River Laboratories),twice daily by intravenous injection, once a week, for three weeks (Days1, 8 and 15), followed by a 10 week treatment-free period. The testantibody was a commercially available mouse anti-AGE antibody raisedagainst carboxymethyl lysine conjugated with keyhole limpet hemocyanin.A control reference of physiological saline was used in the controlanimals.

Mice referred to as “young” were 8 weeks old, while mice referred to as“old” were 88 weeks (±2 days) old. No adverse events were noted from theadministration of the antibody. The different groups of animals used inthe study are shown in Table 1.

TABLE 1 The different groups of animals used in the study Number ofAnimals Dose Level Main Treatment- Group Test (μg/gm/BID/ Study Free No.Material Mice week) Females Females 1 Saline young 0 20 — 2 Saline old 020 20 3 Antibody old 2.5 20 20 4 None old 0 20 pre 5 Antibody old 5.0 2020 — = Not Applicable, Pre = Subset of animals euthanized prior totreatment start for collection of adipose tissue.

p16^(INK4A) mRNA, a marker for senescent cells, was quantified inadipose tissue of the groups by Real Time-qPCR. The results are shown inTable 2. In the table ΔΔCt=ΔCt mean control Group (2)-ΔCt meanexperimental Group (1 or 3 or 5); Fold Expression=2^(−ΔΔCt).

TABLE 2 P16^(INK4a) mRNA quantified in adipose tissue Calculation(unadjusted Group 2 vs Group 1 Group 2 vs Group 3 Group 2 vs Group 5 toGroup 4: 5.59) Group 2 Group 1 Group 2 Group 3 Group 2 Group 5 Mean ΔCt5.79 7.14 5.79 6.09 5.79 7.39 ΔΔCt −1.35 −0.30 −1.60 Fold Expression2.55 1.23 3.03

The table above indicates that untreated old mice (Control Group 2)express 2.55-fold more p16^(ink4a) mRNA than the untreated young mice(Control Group 1), as expected. This was observed when comparing Group 2untreated old mice euthanized at end of recovery Day 85 to Group 1untreated young mice euthanized at end of treatment Day 22. When resultsfrom Group 2 untreated old mice were compared to results from Group 3treated old mice euthanized Day 85, it was observed that p16^(ink4a)mRNA was 1.23-fold higher in Group 2 than in Group 3. Therefore, thelevel of p16^(ink4a) mRNA expression was lower when the old mice weretreated with 2.5 pg/gram/BID/week of antibody.

[85] When results from Group 2 (Control) untreated old mice werecompared to results from Group 5 (5 pg/gram) treated old mice euthanizedDay 22, it was observed that p16^(ink4a) mRNA was 3.03-fold higher inGroup 2 (controls) than in Group 5 (5 μg/gram). This comparisonindicated that the Group 5 animals had lower levels of p16^(ink4a) mRNAexpression when they were treated with 5.0 pg/gram/BID/week, providingp16^(Ink4a) mRNA expression levels comparable to that of the younguntreated mice (Group 1). Unlike Group 3 (2.5 pg/gram) mice that wereeuthanized at end of recovery Day 85, Group 5 mice were euthanized atend of treatment Day 22.

These results indicate the antibody administration resulted in thekilling of senescent cells.

The mass of the gastrocnemius muscle was also measured, to determine theeffect of antibody administration on a classic sign of aging,sarcopenia. The results are shown in Table 3. The results indicate thatadministration of the antibody increased muscle mass as compared tocontrols, but only at the higher dosage of 5.0 μg/gm/BID/week.

TABLE 3 Effect of antibody administration on mass of the gastrocnemiusmuscle Absolute weight of Weight relative Summary Gastrocnemius to bodymass of Group Information Muscle Gastrocnemius Muscle 1 Mean 0.32911.1037 SD 0.0412 0.1473 N 20 20 2 Mean 0.3304 0.7671 SD 0.0371 0.1246 N20 20 3 Mean 0.3410 0.7706 SD 0.0439 0.0971 N 19 19 5 Mean 0.4074 0.9480SD 0.0508 0.2049 N 9 9

These results demonstrate that administration of antibodies that bind toAGEs of a cell resulted in a reduction of cells expressing p16^(ink4a),a biomarker of senescence. The data show that reducing senescent cellsleads directly to an increase in muscle mass in aged mice. These resultsindicate that the loss of muscle mass, a classic sign of sarcopenia, canbe treated by administration of antibodies that bind to AGEs of a cell.

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What is claimed is:
 1. A method of treating or preventing a disease ordisorder associated with cellular senescence, comprising immunizing asubject in need thereof against AGE-modified proteins or peptides of acell, wherein the disease or disorder is selected from the groupconsisting of sarcopenia, inflammation, the onset of cataracts, theonset of loss of adipose tissue, the onset of lordokyphosis, auto-immunedisorders, and neurodegenerative disorders.
 2. A method of treating asubject with a disease or disorder associated with cellular senescence,comprising: administering a first vaccine comprising a first AGEantigen; and administering a second vaccine comprising a second AGEantigen; wherein the second AGE antigen is different from the first AGEantigen and the disease or disorder associated with cellular senescencedoes not comprise sarcopenia.
 3. (canceled)
 4. The method of claim 1,wherein the immunizing comprises administering a vaccine comprising anAGE antigen.
 5. The method of claim 4, wherein the vaccine comprises (a)the AGE antigen, (b) an adjuvant, (c) optionally, a preservative, and(d) optionally, an excipient.
 6. The method of claim 1, wherein thesubject is selected from the group consisting of humans, goats, sheep,cows, horses, dogs and cats.
 7. The method of claim 6, wherein thesubject is a human.
 8. The method of claim 4, wherein the vaccine isadministered in an amount effective to cause the immune system toproduce antibodies to cells having AGE-modified proteins or peptides. 9.The method of claim 1, wherein the subject does not have diabetes. 10.The method of claim 4, wherein the AGE antigen comprises, anAGE-modified protein or peptide selected from the group consisting ofAGE-RNAse, AGE-human hemoglobin, AGE-human serum albumin, AGE-lowdensity lipoprotein, AGE-collagen IV, AGE-antithrombin III,AGE-calmodulin, AGE-insulin, AGE-ceruloplasmin, AGE-collagen,AGE-cathepsin B, AGE-albumin, AGE-crystallin, AGE-plasminogen activator,AGE-endothelial plasma membrane protein, AGE-aldehyde reductase,AGE-transferrin, AGE-fibrin, AGE-copper/zinc SOD, AGE-apo B,AGE-fibronectin, AGE-pancreatic ribose, AGE-apo A-I and II,AGE-hemoglobin, AGE-Na⁺/K⁺-ATPase, AGE-plasminogen, AGE-myelin,AGE-lysozyme, AGE-immunoglobulin, AGE-red cell Glu transport protein,AGE-β-N-acetyl hexokinase, AGE-apo E, AGE-red cell membrane protein,AGE-aldose reductase, AGE-ferritin, AGE-red cell spectrin, AGE-alcoholdehydrogenase, AGE-haptoglobin, AGE-tubulin, AGE-thyroid hormone,AGE-fibrinogen, AGE-β₂-microglobulin, AGE-sorbitol dehydrogenase,AGE-ai-antitrypsin, AGE-carbonate dehydratase, AGE-hexokinase, AGE-apoC-I, AGE-keyhole limpet hemocyanin (AGE-KLH) and mixtures thereof. 11.The method of claim 4, wherein the AGE antigen comprises at least oneprotein or peptide that exhibits AGE modifications selected from thegroup consisting of carboxymethyllysine, carboxyethyllysine,pentosidine, pyrraline, FFI, AFGP, and ALI.
 12. The method of claim 11,wherein the AGE antigen comprises a carboxymethyllysine-modified proteinor peptide.
 13. The method of claim 4, wherein the vaccine is sterile,and the vaccine is in unit dosage form.
 14. The method of claim 4,wherein the vaccine is sterile, and the vaccine is in multidosage form.15-35. (canceled)
 36. The method of claim 1, further comprising testingthe subject to determine if the disease or disorder associated withcellular senescence has been ameliorated, and repeating the immunizing,if necessary.
 37. (canceled)
 38. (canceled)
 39. A method of treating orpreventing atherosclerosis associated with cellular senescence,comprising immunizing a subject in need thereof against AGE-modifiedproteins or peptides of a cell, wherein the immunizing comprisesadministering a vaccine comprising an AGE antigen, and the AGE antigenis not AGE-low density lipoprotein.
 40. The method of claim 39, whereinthe AGE antigen comprises an AGE-modified protein or peptide selectedfrom the group consisting of AGE-RNAse, AGE-human human hemoglobin,AGE-human serum albumin, AGE-collagen IV, AGE-antithrombin III,AGE-calmodulin, AGE-insulin, AGE-ceruloplasmin, AGE-collagen,AGE-cathepsin B, AGE-albumin, AGE-crystallin, AGE-plasminogen activator,AGE-endothelial plasma membrane protein, AGE-aldehyde reductase,AGE-transferrin, AGE-fibrin, AGE-copper/zinc SOD, AGE-apo B,AGE-fibronectin, AGE-pancreatic ribose, AGE-apo A-I and II,AGE-hemoglobin, AGE-Na⁺/K⁺-ATPase, AGE-plasminogen, AGE-myelin,AGE-lysozyme, AGE-immunoglobulin, AGE-red cell Glu transport protein,AGE-β-N-acetyl hexokinase, AGE-apo E, AGE-red cell membrane protein,AGE-aldose reductase, AGE-ferritin, AGE-red cell spectrin, AGE-alcoholdehydrogenase, AGE-haptoglobin, AGE-tubulin, AGE-thyroid hormone,AGE-fibrinogen, AGE-β₂-microglobulin, AGE-sorbitol dehydrogenase,AGE-α₁-antitrypsin, AGE-carbonate dehydratase, AGE-hexokinase, AGE-apoC-l, AGE-keyhole limpet hemocyanin (AGE-KLH) and mixtures thereof. 41.The method of claim 39, wherein the AGE antigen comprises acarboxymethyllysine-modified protein or peptide.
 42. The method of claim39, further comprising testing the subject to determine if theatherosclerosis associated with cellular senescence has beenameliorated, and repeating the immunizing, if necessary.
 43. The methodof claim 2, wherein the first AGE antigen and the second AGE antigeneach independently comprises an AGE-modified protein or peptide selectedfrom the group consisting of AGE-RNAse, AGE-human hemoglobin, AGE-humanserum albumin, AGE-low density lipoprotein, AGE-collagen IV,AGE-antithrombin III, AGE-calmodulin, AGE-insulin, AGE-ceruloplasmin,AGE-collagen, AGE-cathepsin B, AGE-albumin, AGE-crystallin,AGE-plasminogen activator, AGE-endothelial plasma membrane protein,AGE-aldehyde reductase, AGE-transferrin, AGE-fibrin, AGE-copper/zincSOD, AGE-apo B, AGE-fibronectin, AGE-pancreatic ribose, AGE-apo A-I andII, AGE-hemoglobin, AGE-Na⁺K⁺-ATPase, AGE-plasminogen, AGE-myelin,AGE-lysozyme, AGE-immunoglobulin, AGE-red cell Glu transport protein,AGE-6-N-acetyl hexokinase, AGE-apo E, AGE-red cell membrane protein,AGE-aldose reductase, AGE-ferritin, AGE-red cell spectrin, AGE-alcoholdehydrogenase, AGE-haptoglobin, AGE-tubulin, AGE-thyroid hormone,AGE-fibrinogen, AGE-β₂-microglobulin, AGE-sorbitol dehydrogenase,AGE-ai-antitrypsin, AGE-carbonate dehydratase, AGE-hexokinase, AGE-apoC-I, AGE-keyhole limpet hemocyanin (AGE-KLH) and mixtures thereof. 44.The method of claim 2, wherein the first AGE antigen comprises acarboxymethyllysine-modified protein or peptide.